Natural and synthetic nucleosides have important physiologic and pharmacologic activities in humans. Adenosine, for example, is a local signaling molecule with regulatory functions in lipolysis, neurotransmitter release, platelet aggregation, coronary vasodilation, and cardiac contractility (Belardinelli et al., 1989; Jacobson et al., 1990). Nucleoside antimetabolites have therapeutic applications in human neoplastic and viral diseases, including leukemias and AIDS (Perigaud et al., 1992; Handschumacher and Cheng, 1993).
Most nucleoside drugs act intracellularly, after anabolic phosphorylation, by interfering, either directly or indirectly, with DNA synthesis. For those nucleosides that are hydrophilic, mediated transport systems (NT processes) are required for passage across the plasma membrane. In experimental systems, there is evidence that the activity of NT FNT The abbreviations used are: AIDS, acquired immunodeficiency syndrome; HIV, human immunodeficiency virus; NBMPR, nitrobenzylthioinosine (6-[(4-nitrobenzyl)thio]-9-p-D-ribofuranosylpurine); AZT, 3'-azido-3'-deoxythymidine; ddC, 2',3'-dideoxycytidine; ddG, 2',3'-dideoxyguanosine; ddI, 2',3'-dideoxyinosine; bp, base pair(s); kb, kilobase(s). cDNA, complementary DNA; DAPI, 4',6-diamidin-2-phenylindol-dihydrochloride; ddC, 2',3'-dideoxycytidine; FISH fluorescence in situ hybridization; avidin-FITC, avidin-fluorescein isothiocyanate; HIV, human immunodeficiency virus; kb, kilobase(s); NT, nucleoside transporter; PAC, P1-derived artificial chromosome; poly(A).sup.+, polyadenylated; PCR, polymerase chain reaction; RT-PCR, reverse manscriptase polymerase chain reaction; CNT1 and CNT1 both represent concentrative nucleoside transporter 1.
processes can be an important determinant of pharmacologic action of cytotoxic nucleoside drugs. For example, cultured cells made incapable of transporting nucleosides by genetic mutations or treatment with NT inhibitors exhibit low levels of uptake of adenosine and other endogenous nucleosides and are resistant to a variety of nucleoside analogs with anticancer activity The permeant selectivities and mechanisms regulating distribution and expression of NT processes are important factors to be considered in the design of nucleoside analogs as therapeutic agents in human diseases.
Both equilibrative and Na.sup.+ -dependent nucleoside transport mechanisms are present in mammalian cells. In human erythrocytes, transport of purine and pyrimidine nucleosides is equilibrative (Na.sup.+ -independent) and inhibited by namomolar concentrations of NBMPR (Young and Jarvis, 1983; Paterson et al, 1983). The erythrocyte transporter, an integral membrane glycoprotein of apparent M.sub.T 55,000 (Wu et al 1983), has been purified to apparent homogeneity by a combination of ion-exchange and immunoaffinity chromatography (Kwong et al 1988). Functionally and structurally similar equilibrative nucleoside transporters (designated es) are widely distributed in mammalian cells and tissues (Paterson et al, 1991; Kwong et al, 1993)). In addition, some mammalian cells and tissues (Paterson et al, 1991) possess Na.sup.+ -independent nucleoside transport systems with low (micromolar) sensitivity to inhibition by NBMPR (designated ei). The molecular properties of ei transporters are unknown.
Na.sup.+ -dependent nucleoside transport systems have been demonstrated in a variety of cell types, including intestinal (Vijayalakshimi and Belt, 1988; Jarvis, 1989; Roden et al, 1991), renal epithelia (Lee et al, 1990; Williams and Jarvis, 1991; Gutierrez and Giacomini, 1993), and choroid plexus (Wu et al, 1992), liver (Che et al., 1992); splenocytes (Plagemann et al., 1990), macrophages (Plagemnann, 1991)and leukemia cells (Belt et al., 1993, Paterson et al. 1993). Active, Na.sup.+ -linked NT processes are present in intestinal (Betcher et al., 1990; Vijayalakshmi and Belt, 1988) and renal epithelia (Gutierrez and Giacomini 1993; Le Hir and Dubach, 1984; Williams et al., 1989), choroid plexus (Wu et al., 1994), liver (Che et al., 1992), splenocytes (Plagemann et al., 1990), macrophages (Plagemann, 1991) and leukemic cells (Belt et al. 1993; Paterson et al., 1993). The two principal Na.sup.+ -dependent NT subtypes, designated N1 (or cif) and N2 (or cit), have complementary and overlapping selectivities for purine nucleosides and uridine (N1/cif) and pyrimidine nucleosides and adenosine (N2/cit). N1/cif and N2/cit NTs have been found in tissues and cells of rat, rabbit, murine and bovine origin (Cass, 1995). A third class of Na.sup.+ -dependent NTs, designated N3 (or cib), found to date only in rat jejunum (Huang et al., 1993), rabbit choroid plexus (Wu et al., 1994) and human leukemic cells (Belt et al., 1993), has the ability to transport a wide range of both purine and pyrimidine nucleosides. A human kidney N2/cit-like process that is selective for guanosine in addition to pyrimidine nucleosides and adenosine has been given the designation N4 (Gutierrez and Giacomini, 1993; Gutierrez and Giacomini, 1994). Finally, a Na.sup.+ -dependent NT activity that is inhibited by NBMPR and designated N5 (or cs) has been observed in freshly isolated human leukemic cells (Paterson et al., 1993). It is not known from the current literature if human cells express the N1/cif and N2/cit NT subtypes (Cass, 1995).
It has been demonstrated that Xenopus oocytes express Na.sup.+ -dependent nucleoside transport activity after microinjection of poly (A).sup.+ RNA from rat jejunum (Huang et al, 1993).